The long-term objective of this proposal is to understand the functions of protein domains in cell adhesion and signaling. Although many somatic cell adhesion and signaling events are understood in impressive detail, the molecular basis of species-specific gamete interactions in mammals is unclear. The recent discovery of a sperm membrane protein (zonadhesin) that binds directly and in a species-specific manner to the egg extracellular matrix (zona pellucida, ZP) suggests that this protein functions in sperm-egg adhesion. The cDNA of zonadhesin encodes a precursor molecule comprising seven extracellular domains, including a mucin domain and five tandem domains homologous to the D-domains of von Willebrand factor. The proposed experiments will investigate aspects of zonadhesin's activity, domain structure, and biogenesis that are relevant to the hypothesis that this protein mediates species-specific adhesion of spermatozoa to the ZP. The specific aims are: 1) to complete a definitive ultrastructural localization of zonadhesin; 2) to discover the functions of native zonadhesin and its various individual domains in gamete interactions; 3) to characterize zonadhesin's biogenesis and activation during spermiogenesis and capacitation; and 4) to test whether zonadhesin couples to known cellular signaling cascades of spermatozoa, including G-proteins, adenylyl cyclase, and protein kinass. Aim 1 will be achieved by subcellular fractionation, fluorescence staining of living sperm cells, and immunelectron microscopy. Aim 2 will be achieved by characterizing direct binding of zonadhesin domains to native ZP, and by sperm-ZP adhesion-inhibiting and adhesion-promoting experiments. Aim 3 will be completed using immunochemical methods. Aim 4 will test the activities of zonadhesin's putative cytoplasmic tail in various cell signaling assays. Results of these experiments will be most directly relevant to diagnosis and treatment of infertility, and development of new contraceptives. By adding to general knowledge of cell-cell interactions, they may also lead to a better understanding of other medical conditions such as development problems, cancer, blood disorders, and neurological diseases.